The antibiotic minimycin

ABSTRACT

A novel antibiotic Minimycin characterized by a needle or pillar-like crystal form which is soluble in water and lower alcohols, but which is insoluble in nonpolar organic solvents, has been shown to be active against Staphylococcus and Colibacillus, which are ordinarily resistant to other antibiotics.

United States Patent Shirato et al.

[ Aug. 28, 1973 THE ANTIBIOTIC MINIMYCIN Inventors: Shiro Shirato; Junsaku Nagatsu;

Mitsuo Shibuya, all of Tokyo; Yoko Kusakabe, Tokorozawa, all of Japan Kaken Kagaku Kabushlki Kaisha, Tokyo, Japan Filed: Sept. 4, 1970 Appl. No.: 69,782

Assignee:

Foreign Application Priority Data Sept. 5, 1969 Japan 44/159949 US. Cl. 260/209 R, 195/80, 424/180 Int. Cl. C07c 69/20 Field of Search 260/209, 210 AB [56] References Cited UNITED STATES PATENTS 3,480,613 ll/l969 Walton 260/209 R Primary Examiner-Lewis Gotts Assistant Examiner-Johnnie R. Brown Attorney-Oblon, Fisher and Spivak [5 7] ABSTRACT 1 Claim, 2 Drawing Figures LR. Absorption Spectrum of MinimycimA PATENTED M1928 ms SHEET 1 0F 2 BY (936m. 554

mvmvmus Sumo SHIRATO M ATTORNEYS LR. Absorption Spectrum of Minimycin3 KB PATENTED AUG 28 I973 SHEUZBFZ BUD SM 3000 WAVE NUMBER CH ATTORNEYS THE ANTIBIOTIC MINIMYCIN BACKGROUND OF THE INVENTION This invention relates to "the novel needle or pillar crystal fonn Minimycin and, more particularly, to Minimycin which is produced by culturing a novel microorganism.

DESCRIPTION OF THE PRIOR ART It has been known that the genus Streptomyces includes various species, some of which will produce antibiotics. However, a need exists for novel antibiotics which are effective against Staphylococcus and Calibacillus, which are resistant to penicillin, Streptomycin, Kanamycin, Tetracycline, Erythromycin, Chloramphenicol and Sulfonamide.

SUMMARY OF THE INVENTION Accordingly, it is one object of this invention to provide a novel antibiotic which is effective against Staphylococcus and Calibacillus of the type which are resistant to Penicillin, Streptomycin, Kanamycin, Tetracycline, Erythromycin, Chloramphenicol and Sulfonamide.

DETAILED DESCRIPTION OF THE INVENTION The novel antibiotic Minimycin is produced by culturing Strepromyces sp 80,432, a species of Actinomycetes. Streptomyces sp 80,432, has been isolated from a soil collection obtained from Nyugawa, Ehime Prefecture, and has been deposited in the Japanese Agency of Industrial Science, Fermentation Research Institute under the serial number of PERM-P 364. A deposit of this bacterium has been made with the American Type Culture Collection, Rockville, Maryland, and has been assigned ATCC No. 21589. Streptomyces sp 80,432 is characterized by similar properties as genus Streptomyces is Actinomycetes. Its cultural and physiological characteristics and carbon source utilization are shown in Tables 1, 2 and 3.

The morphological characteristics of Streptomyces sp 80,432 are as follows:

Vegetative mycelia never fragment into bacillary or coccorid elements when branched finely.

The sporophores are formed as compact spirals, primitive spirals or loops, but are sometimes straight.

The compact and primitive spirals of sporophores are occasionally observed to be spherical at the termination of the culture.

The structure of the spore surface is smooth, and the size of the spore is 1.0 1.2 X 0.8 yr.

Physiologically, the Streptomyces sp 80,432 is of the chromogenic type and is characterized by hygroscopic properties under specific cultural conditions.

TABLE 1 Cultural characteristics of Slrepromycer .rp 80,432 (observed at 27C for 20 days) Aerial Soluble Growth Mycelium pigment Nurient Poor and thin; colonial- None None agar yellow (2 ga) or Russet orange (4 nc) Glucose Good and thick; None None nutri nt wrinkles; Light ivory agar (2 ca) to Russet orange (4 nc) Trypton Poor or Moderate; None Slightly yeast Light ivory (2 ca) to dark extract pearl pink (3 ca) brown ag Potato Good and thick; Quite scant and None glucose wrinkles and rapid: white: sometimes agar colonial yellow (2 qa) none formed or honey gold (21 c) Yezm Good and thick and Abundant; pearl gray None or extractwrinkled; Light (near grays 13 ch); light Malt amber (3 ie) to sometimes partially brown extract amber (3 nc) hygroscopic agar Oatmeal Good and thin; white Abundant and None agar or yellowish powdery; pearl gray White (near grays 13 ch) or beige gray (near grays 3 ih); sometimes partially hygroscopic 590F068 Speedy, flat and thin; None None "mate cream (1% ca) agar Glycerol Poor and thin; light None None nitrate ivory (2 ca) agar Starch Flat and good; white Abundant and None agar to light apricot (4 ea) powdery; pearl gray or light ivory (2 ca) (near gray 13 ch) or beige gray (near gray 3 ih); sometimes partially hygroscopic; some times scant and white Glucose Poor and thin; Medium or poor; None asparagine colorless to light white or pearl gray agar ivory(2 ca) (near grays 13 ch); natural (near grays 3 dc) Glycerol- Good and wrinkles; None or quite scant; None or Ca-malate white to light ivory white slightly agar (2 ie) dissolving Camalate Tyrosine 'Good and thick and Surrounding of Whole yeast wrinkled; mustard tan growth part extract (2 or dull gold of agar agar 8) black pepmne Medium or poor ancl None v Whole on agar thin;=Brike maize part (3 le) or Brite (3 ie) ofagap black Potato Good and thick and None or quite scant; Black plug wrinkled; Golden white brown (3 pi) L fi Poor and thin; Brown None Slightly medium dark I brown Egg Good and thick; dark None D -k medium brown brown Glucose Poor on surface as thin None None nitrate membrane solution Physiological Reactions of Strepromyce: sp 80,432 Test Response Melanin formation:

i im 1 P) Tyrosinase' reaction Hydrogen sulfide production Nitrate reduction Liflg'faction of gelatin Coagulation Cellulose utilimtion Starch hydrolysis Temperature range (pH 7.0) pH range Positive (2-3 days) Positive (2-3 days) Doubtful (14 days) Positive Positive Positive Negative PNositive eganve Negative Positive No growth at 15C and 50C Growth at pH 4.5 and pH 9.5

' Melezitose sources by esponse Trehalose Rhamnose Mannose Xylose Salicin lnulin lnositol Dulsitol Adonitol Mannitol Sorbitol -H-: strongly positive doubtful positive positive negative The culture of Streptomyces sp 80,432, appears yellowish in coler when grown in a proteinaceous media. The same color is also observed when grown in a synthetic media.

In order to produce Minimycin by culturing the Streptomyces .rp 80,432, conventional culturing methods of Actinomycetes, can be employed; on an industrial scale, aerobic culturing in a tank is preferable.

The accumulation of Minimycin in the media will be maximized by culturing at 20-32C., preferably 25-29C, for about 2-4 days. The cultured media is then filtered and the Minimycin is isolated and purified. Although Minimcyin can be absorbed by activated carbon over a wide pH range, it is preferable to absorb it at a pH of 2.0 from the point of view of stability and adsorption. After adsorption, the Minimycincontaining activated carbon is adsorbed, filtered and washed with water. The Minimycin is eluted. from the activated carbon by treatment with a hydrophilic solvent such as 50 percent acetone-water. The solvent is distilled from the Minimycin solution in vacuo and Minimycin is isolated by chromatography using an activated carbon column. The resulting antibiotic active fractions are collected and distilled in vacuo to obtain a pure concentrated solution. The solution is filtered in a column containing a gel filter material and is eluted with water and fractionated. The antibiotic active fractions are collected and freeze-dried. A colorless needle crystalline form of Minimycin can be obtained by recrystallizing the resulting white power from water. In order to produce colorless pillar crystalline from Minimycin the resulting white powder is recrystallized from an appropriate organic solvent such as ethanol or acetone.

The Minimycin has the'following properties:

1. Chemical structurez 2. Colorless needle or pillar crystalline forms melt ing point is 161C. 166C. (Decomposition: needle 166C; pillar 161C).

3. Elementary analysis:

C: 44.15 percent; H: 4.62 percent; N: 5.84 percent; and O: 45.39 percent MW 245 (vapor pressure); Molecular formula C,H NO,

4. Solubility:

Soluble in water; soluble in lower alkyl alcohols; insoluble or less soluble in organic solvents (lyophilic).

5. Infrared spectrum:

Infrared spectrum of the needle crystalline type of Minimycin is shown in FIG. I and that of pillar crystalline type of Minimycin is shown in FlG. 2.

6. Ultraviolet absorption spectrum: 230 my. shoulder (1 percent 11 0). 7. (0th, 18.0 (C1, water). 8. Rf value on paper chromatography: Rf values in various elutes are as follows:

Water-saturated butanol 0.25 Butanol-Acetic acid-water (4:1:2) 0.49 Ethanol-Water (4:1) 0.67 Pr anol-pyridine-acetic acid-water 0.85

op (1S:10:3:12)

9. Rf value on thin layer chromatography:

Rf values on silica gel thin layer are as follows: Water-Saturated butanol 0.42

Butanol-Acetic acid-water (4:1 :2) 0.57 Ethanol-Water (4:1) 0.75 Chloroform-methano1-17% ammonia 0.88

(2:1:1 upper layer of mixture) 10. Color reaction: I

Discoloring potassium permanganate;

Negative for ninhydrin reaction, anisaldehyde reaction, 2,3,5-triphenyltetrazoliumchloride reaction, El-

' son-Morgan reaction, Biuret reaction and Mohlish (yellow ring) reaction.

1 1. Heat stability:

Little decreasing of activity occurs by heating at C for 10 minutes in pH 5 and pH 2. However, activity is decreased in pH 7 and pH 9 at 100C for 10 minutes. No decrease in activity is found by heating at 50C for 300 minutes at pH 5 and pH 2; however, a slight decrease of activity is found at pH 7 and pH 9. No decrease is found at 27C for 24 hours at pH 2 8.

l2. Antibiotical spectrum:

The obstruct areas resulted by the agar-disc difiusion method are stated, respectively, in the following Table.

TABLE 4 Staphylococcus E. Coli Minimycin A sensitive durable sensitive durable meg/ml strain strain strain strain By the term durable strain of Staphylococcus is meant a strain which is resistant to Penicillin, Streptomycin, Kanamycin, Chloraphenicol, Tetracycline, Erythromycin and Sulfonamide. By the term durable strain of Colibacillus is meant a strain which is resistant to Streptomycin, Kanamycin, Chloraphenicol, Tetracycline and Sulfonamide.

l3. Toxicity:

Test mice were given intravenous injection of 100 mg/kg weight of the antibiotic. Half the mice died within 24 hours, and all of the mice died within 72 hours. However, by intravenous injection of 50 mglkg,

weight, all of the mice survived.

A further understanding of this invention can be obtained by reference to the following specific examples, which are provided herein for the purpose of illustration. In these examples, all percentages are intended to be referred to as percent by weight, unless otherwise stated.

EXAMPLE 1 100 liters of media containing 4.0 percent of grape sugar, 2.0 percent of soybean flour, 1.5 percent of wheat embryo bud, 0.4 percent of dried brewers yeast, 0.2 percent of salt, 0.2 percent of potassium chloride and 0.2 percent of calcium carbonate, is placed into a 200 liter tank. Streptomyces sp 80,432 is inoculated and cultured at 27C for 72 hours by aerobic culturing. The product is filtered and the pH of the filtrate is adjusted to 2.0 by adding hydrochloride. 3 percent of activated carbon based on the weight of filtrate is added and the filtrate stirred. The same amount of diatomaceous earth is then added to the filtrate. The mixture is filtrated and the residuum is washed and mixed with a 50 percent acetone-water solution. It is then filtered and condensed in vacuo to obtain about liters of concentrate. The concentrate is past downward from the top of the column packed with 6 liters of activated carbon, and then the column is washed with water and eluted with 20 percent acetone-water. The elute fractions having the strongest antibiotical activity are collected and condensed in vacuo to produce the pure concentrate. The pure concentrate is past downwardly from the top of the column packed with dilica (Sephadex 6-10) and then is eluted with water. The antibiotical activity of each elute fraction is tested by disc-plate diffusion methods and the fractions having the highest antibiotical activity are collected, and freeze-dried to obtain a white powder of Minimycin.

EXAMPLE 2 The white powder of Minimycin obtained in Example 1 is recrystallized from water to yield 10 g of pure Minimycin having a colorless needle crystalline form. The melting point is 166C (decomposition).

EXAMPLE 3 The white powder of Minimycin obtained in Example 1 is recrystallized from ethanol to yeild 10 g of pure Minimycin having a colorless pillar crystalline form. The melting point is 161C (decomposition).

The results of the pharmacological test of Minimycin show that LDSO (ip) 50 mg/kg and Minimycin has high antibiotical activity especially for strains durable to other known antibiotics.

Having now fully described the invention, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention. Accordingly,

What is claimed is:

1. An antibiotic, Minimycin, having the formula: 

